Microparticles with adsorbent surfaces, methods of making same, and uses thereof

ABSTRACT

Microparticles with adsorbent surfaces, methods of making such microparticles, and uses thereof, are disclosed. The microparticles comprise a polymer, such as a poly(α-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a polyanhydride, and the like, and are formed using cationic, anionic, or nonionic detergents. The surface of the microparticles efficiently adsorb biologically active macromolecules, such as DNA, polypeptides, antigens, and adjuvants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/581,772, filed Jun. 15, 2000, which is a 371 of InternationalApplication No. PCT/US99/17308, filed Jul. 29, 1999, which is acontinuation-in-part of U.S. patent application Ser. No. 09/285,855,filed Apr. 2, 1999, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/124,533, filed Jul. 29, 1998, which is acontinuation-in-part of U.S. patent application Ser. No. 09/015,652,filed Jan. 29, 1998, which claims the benefit of U.S. ProvisionalApplication Ser. Nos. 60/036,316, filed Jan. 30, 1997 and 60/069,749,filed Dec. 16, 1997. Each of the above applications are incorporatedherein by reference in their entireties.

TECHNICAL FIELD

The present invention relates generally to pharmaceutical compositions.In particular, the invention relates to microparticles with adsorbentsurfaces, methods for preparing such microparticles, and uses thereof.Additionally, the invention relates to compositions comprisingbiodegradable microparticles wherein biologically active agents, such astherapeutic polynucleotides, polypeptides, antigens, and adjuvants, areadsorbed on the surface of the microparticles.

BACKGROUND

Particulate carriers have been used in order to achieve controlled,parenteral delivery of therapeutic compounds. Such carriers are designedto maintain the active agent in the delivery system for an extendedperiod of time. Examples of particulate carriers include those derivedfrom polymethyl methacrylate polymers, as well as microparticles derivedfrom poly(lactides) (see, e.g., U.S. Pat. No. 3,773,919),poly(lactide-co-glycolides), known as PLG (see, e.g., U.S. Pat. No.4,767,628) and polyethylene glycol, known as PEG (see, e.g., U.S. Pat.No. 5,648,095). Polymethyl methacrylate polymers are nondegradable whilePLG particles biodegrade by random nonenzymatic hydrolysis of esterbonds to lactic and glycolic acids which are excreted along normalmetabolic pathways.

For example, U.S. Pat. No. 5,648,095 describes the use of microsphereswith encapsulated pharmaceuticals as drug delivery systems for nasal,oral, pulmonary and oral delivery. Slow-release formulations containingvarious polypeptide growth factors have also been described. See, e.g.,International Publication No. WO 94/12158, U.S. Pat. No. 5,134,122 andInternational Publication No. WO 96/37216.

Fattal et al., Journal of Controlled Release 53:137-143 (1998) describesnanoparticles prepared from polyalkylcyanoacrylates (PACA) havingadsorbed oligonucleotides.

Particulate carriers have also been used with adsorbed or entrappedantigens in attempts to elicit adequate immune responses. Such carrierspresent multiple copies of a selected antigen to the immune system andpromote trapping and retention of antigens in local lymph nodes. Theparticles can be phagocytosed by macrophages and can enhance antigenpresentation through cytokine release. For example, commonly owned,co-pending application Ser. No. 09/015,652, filed Jan. 29, 1998,describes the use of antigen-adsorbed and antigen-encapsulatedmicroparticles to stimulate cell-mediated immunological responses, aswell as methods of making the microparticles.

In commonly owned provisional patent Application 60/036,316, forexample, a method of forming microparticles is disclosed which comprisescombining a polymer with an organic solvent, then adding an emulsionstabilizer, such as polyvinyl alcohol (PVA), then evaporating theorganic solvent, thereby forming microparticles. The surface of themicroparticles comprises the polymer and the stabilizer. Macromoleculessuch as DNA, polypeptides, and antigens may then be adsorbed on thosesurfaces.

It has also been shown that cationic lipid-based emulsions may be usedas gene carriers. See, e.g., Yi et al., Cationic Lipid Emulsion; a NovelNon-Viral, and Non-Liposomal Gene Delivery System, Proc. Int'l. Symp.Control. Rel. Bioact. Mater., 24:653-654 (1997); Kim et al., In VivoGene Transfer Using Cationic Lipid Emulsion-Mediated Gene DeliverySystem by Intra Nasal Administration, Proc. Int'l. Symp. Control. Rel.Bioact. Mater., 25:344-345 (1998); Kim et al., In Vitro and In Vivo GeneDelivery Using Cationic Lipid Emulsion, Proc. Int'l. Symp. Control. Rel.Bioact. Mater., 26, #5438 (1999).

While antigen-adsorbed PLG microparticles offer significant advantagesover other more toxic systems, adsorption of biologically active agentsto the microparticle surface can be problematic. For example, it isoften difficult or impossible to adsorb charged or bulky biologicallyactive agents, such as polynucleotides, large polypeptides, and thelike, to the microparticle surface. Thus, there is a continued need forflexible delivery systems for such agents and, particularly for drugsthat are highly sensitive and difficult to formulate.

SUMMARY OF THE INVENTION

The inventors herein have invented a method of forming microparticleswith adsorbent surfaces capable of adsorbing a wide variety ofmacromolecules. The microparticles are comprised of both a polymer and adetergent. The microparticles of the present invention adsorb suchmacromolecules more efficiently than other microparticles currentlyavailable.

The microparticles are derived from a polymer, such as a poly(α-hydroxyacid), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester,a polyanhydride, a PACA, a polycyanoacrylate, and the like, and areformed with detergents, such as cationic, anionic, or nonionicdetergents, which detergents may be used in combination. Additionally,the inventors have discovered that these microparticles yield improvedadsorption of viral antigens, and provide for superior immune responses,as compared to microparticles formed by a process using only PVA. Whilemicroparticles made using only PVA may adsorb some macromolecules, themicroparticles of the present invention using other detergents alone, incombination, or in combination with PVA, adsorb a wide variety ofmacromolecules. Accordingly, then, the invention is primarily directedto such microparticles, as well as to processes for producing the sameand methods of using the microparticles.

In one embodiment, the invention is directed to a microparticle with anadsorbent surface, wherein the microparticle comprises a polymerselected from the group consisting of a poly(α-hydroxy acid), apolyhydroxy butyric acid, a polycaprolactone, a polyorthoester, apolyanhydride, and a polycyanoacrylate.

In another embodiment, the invention is directed to such microparticleswhich further comprise a selected macromolecule adsorbed on themicroparticle's surface, such as a pharmaceutical, a polynucleotide, apolypeptide, a protein, a hormone, an enzyme, a transcription ortranslation mediator, an intermediate in a metabolic pathway, animmunomodulator, an antigen, an adjuvant, or combinations thereof, andthe like.

In another embodiment, the invention is directed to a microparticlecomposition comprising a selected macromolecule adsorbed to amicroparticle of the invention and a pharmaceutically acceptableexcipient.

In another embodiment, the invention is directed to a microparticlecomprising a biodegradable polymer and an ionic surfactant.

In another embodiment, the invention is directed to a method ofproducing a microparticle having an adsorbent surface, the methodcomprising:

-   -   (a) combining a polymer solution comprising a polymer selected        from the group consisting of a poly(α-hydroxy acid), a        polyhydroxy butyric acid, a polycaprolactone, a polyorthoester,        a polyanhydride, and a polycyanoacrylate, wherein the polymer is        present at a concentration of about 1% to about 30% in an        organic solvent;    -   and an anionic, cationic, or nonionic detergent to the polymer        solution, wherein the detergent is present at a ratio of 0.001        to 10 (w/w) detergent to polymer, to form a polymer/detergent        mixture;    -   (b) dispersing the polymer/detergent mixture;    -   (c) removing the organic solvent; and    -   (d) recovering the microparticle.

Preferably, the polymer/detergent mixture is emulsfied to form anemulsion prior to removing the organic solvent.

In another embodiment, the invention is directed to a microparticleproduced by the above described methods.

In another embodiment, the invention is directed to a method ofproducing a microparticle with an adsorbed macromolecule comprising:

-   -   (a) combining a polymer solution comprising        poly(D,L-lactide-co-glycolide), wherein the polymer is present        at a concentration of about 3% to about 10% in an organic        solvent;    -   and an anionic, cationic, or nonionic detergent, wherein the        detergent is present at a ratio of 0.001 to 10 (w/w) detergent        to polymer, to form a polymer/detergent mixture;    -   (b) dispersing the polymer/detergent mixture;    -   (c) removing the organic solvent from the emulsion;    -   (d) recovering the microparticle; and    -   (e) adsorbing a macromolecule to the surface of the        microparticle, wherein the macromolecule is selected from the        group consisting of a pharmaceutical, a polynucleotide, a        polypeptide, a hormone, an enzyme, a transcription or        translation mediator, an intermediate in a metabolic pathway, an        immunomodulator, an antigen, an adjuvant, and combinations        thereof. Preferably, the polymer/detergent mixture is emulsfied        to form an emulsion prior to removing the organic solvent. In        another embodiment, the invention is directed to a microparticle        with an adsorbed macromolecule produced by the above described        method.

In another embodiment, the invention is directed to a method ofproducing an adsorbent microparticle composition comprising combining anadsorbent microparticle having a macromolecule adsorbed on the surfacethereof and a pharmaceutically acceptable excipient.

In yet another embodiment, the invention is directed to a method ofdelivering a macromolecule to a vertebrate subject which comprisesadministering to a vertebrate subject the composition above.

In an additional embodiment, the invention is directed to a method foreliciting a cellular immune response in a vertebrate subject comprisingadministering to a vertebrate subject a therapeutically effective amountof a selected macromolecule adsorbed to a microparticle of theinvention.

In another embodiment, the invention is directed to a method ofimmunization which comprises administering to a vertebrate subject atherapeutically effective amount of the microparticle composition above.The composition may optionally contain unbound macromolecules, and alsomay optionally contain adjuvants, including aluminum salts such asaluminum phosphate.

In a preferred embodiment, the microparticles are formed from apoly(α-hydroxy acid); more preferably, a poly(D,L-lactide-co-glycolide);and most preferably, a poly(D,L-lactide-co-glycolide).

In a preferred embodiment, the microparticles are for use in diagnosisof a disease.

In a preferred embodiment, the microparticles are for use in treatmentof a disease.

In a preferred embodiment, the microparticles are for use in a vaccine.

In a preferred embodiment, the microparticles are for use in raising animmune response.

Each of the nonexhaustive previously described adsorbent microparticlesmay optionally also have macromolecules entrapped within them.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, polymer chemistry,biochemistry, molecular biology, immunology and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton,Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowickand N. Kaplan, eds., Academic Press, Inc.); Handbook of ExperimentalImmunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986,Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Handbook of Surface andColloidal Chemistry (Birdi, K. S., ed, CRC Press, 1997) andSeymour/Carraher=s Polymer Chemistry (4th edition, Marcel Dekker Inc.,1996).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus, for example, the term Aamicroparticle@ refers to one or more microparticles, and the like.

A. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The term “microparticle” as used herein, refers to a particle of about100 nm to about 150 μm in diameter, more preferably about 200 nm toabout 30 μm in diameter, and most preferably about 500 nm to about 10 μmin diameter. Preferably, the microparticle will be of a diameter thatpermits parenteral or mucosal administration without occluding needlesand capillaries. Microparticle size is readily determined by techniqueswell known in the art, such as photon correlation spectroscopy, laserdiffractometry and/or scanning electron microscopy.

Microparticles for use herein will be formed from materials that aresterilizable, non-toxic and biodegradable. Such materials include,without limitation, poly(α-hydroxy acid), polyhydroxybutyric acid,polycaprolactone, polyorthoester, polyanhydride, PACA, andpolycyanoacrylate. Preferably, microparticles for use with the presentinvention are derived from a poly(α-hydroxy acid), in particular, from apoly(lactide) (“PLA”) or a copolymer of D,L-lactide and glycolide orglycolic acid, such as a poly(D,L-lactide-co-glycolide) (“PLG” or“PLGA”), or a copolymer of D,L-lactide and caprolactone. Themicroparticles may be derived from any of various polymeric startingmaterials which have a variety of molecular weights and, in the case ofthe copolymers such as PLG, a variety of lactide:glycolide ratios, theselection of which will be largely a matter of choice, depending in parton the coadministered macromolecule. These parameters are discussed morefully below.

The term “detergent” as used herein includes surfactants and emulsionstabilizers. Anionic detergents include, but are not limited to, SDS,SLS, sulphated fatty alcohols, and the like. Cationic detergentsinclude, but are not limited to, cetrimide (CTAB), benzalkoniumchloride, DDA (dimethyl dioctodecyl ammonium bromide), DOTAP, and thelike. Nonionic detergents include, but are not limited to, sorbitanesters, polysorbates, polyoxyethylated glycol monoethers,polyoxyethylated alkyl phenols, poloxamers, and the like.

The term “net positive charge” as used herein, means that the charge onthe surface of the microparticle is more positive than the charge on thesurface of a corresponding microparticle made using PVA. Likewise, theterm Anet negative charge@ as used herein, means that the charge on thesurface of the microparticle is more negative than the charge on thesurface of a corresponding microparticle made using PVA. Net charge canbe assessed by comparing the zeta potential (also known aselectrokinetic potential) of the microparticle made using a cationic oranionic detergent with a corresponding microparticle made using PVA.Thus, a microparticle surface having a Anet positive charge@ will have azeta potential greater than the zeta potential of the surface of amicroparticle made using PVA and a microparticle having a Anet negativecharge@ will have a zeta potential less than the zeta potential of thesurface of a microparticle made using PVA. As is apparent, the netcharges for the microparticles of the invention are calculated relativeto the zeta potential of a corresponding PVA microparticle.

The term Azeta potential@ as used herein, refers to the electricalpotential that exists across the interface of all solids and liquids,i.e., the potential across the diffuse layer of ions surrounding acharged colloidal particle. Zeta potential can be calculated fromelectrophoretic mobilities, i.e., the rates at which colloidal particlestravel between charged electrodes placed in contact with the substanceto be measured, using techniques well known in the art.

The term Amacromolecule,@ as used herein, refers to, without limitation,a pharmaceutical, a polynucleotide, a polypeptide, a hormone, an enzyme,a transcription or translation mediator, an intermediate in a metabolicpathway, an immunomodulator, an antigen, an adjuvant, or combinationsthereof. Particular macromolecules for use with the present inventionare described in more detail below.

The term Apharmaceutical@ refers to biologically active compounds suchas antibiotics, antiviral agents, growth factors, hormones, and thelike, discussed in more detail below.

A “polynucleotide” is a nucleic acid molecule which encodes abiologically active (e.g., immunogenic or therapeutic) protein orpolypeptide. Depending on the nature of the polypeptide encoded by thepolynucleotide, a polynucleotide can include as little as 10nucleotides, e.g., where the polynucleotide encodes an antigen.Furthermore, a “polynucleotide” can include both double- andsingle-stranded sequences and refers to, but is not limited to, cDNAfrom viral, procaryotic or eucaryotic mRNA, genomic RNA and DNAsequences from viral (e.g. RNA and DNA viruses and retroviruses) orprocaryotic DNA, and especially synthetic DNA sequences. The term alsocaptures sequences that include any of the known base analogs of DNA andRNA, and includes modifications, such as deletions, additions andsubstitutions (generally conservative in nature), to the nativesequence, so long as the nucleic acid molecule encodes a therapeutic orantigenic protein. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts which produce the antigens.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also include modifications,such as deletions, additions and substitutions (generally conservativein nature), to the native sequence, so long as the protein maintains theability to elicit an immunological response or have a therapeutic effecton a subject to which the protein is administered.

By “antigen” is meant a molecule which contains one or more epitopescapable of stimulating a host's immune system to make a cellularantigen-specific immune response when the antigen is presented inaccordance with the present invention, or a humoral antibody response.An antigen may be capable of eliciting a cellular or humoral response byitself or when present in combination with another molecule. Normally,an epitope will include between about 3-15, generally about 5-15, aminoacids. Epitopes of a given protein can be identified using any number ofepitope mapping techniques, well known in the art. See, e.g., EpitopeMapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E.Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linearepitopes may be determined by e.g., concurrently synthesizing largenumbers of peptides on solid supports, the peptides corresponding toportions of the protein molecule, and reacting the peptides withantibodies while the peptides are still attached to the supports. Suchtechniques are known in the art and described in, e.g., U.S. Pat. No.4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002;Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated hereinby reference in their entireties. Similarly, conformational epitopes arereadily identified by determining spatial conformation of amino acidssuch as by, e.g., x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols, supra.

The term Aantigen@ as used herein denotes both subunit antigens, i.e.,antigens which are separate and discrete from a whole organism withwhich the antigen is associated in nature, as well as killed, attenuatedor inactivated bacteria, viruses, parasites or other microbes.Antibodies such as anti-idiotype antibodies, or fragments thereof, andsynthetic peptide mimotopes, which can mimic an antigen or antigenicdeterminant, are also captured under the definition of antigen as usedherein. Similarly, an oligonucleotide or polynucleotide which expressesa therapeutic or immunogenic protein, or antigenic determinant in vivo,such as in gene therapy and nucleic acid immunization applications, isalso included in the definition of antigen herein.

Further, for purposes of the present invention, antigens can be derivedfrom any of several known viruses, bacteria, parasites and fungi, aswell as any of the various tumor antigens. Furthermore, for purposes ofthe present invention, an “antigen” refers to a protein which includesmodifications, such as deletions, additions and substitutions (generallyconservative in nature), to the native sequence, so long as the proteinmaintains the ability to elicit an immunological response. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe antigens.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto molecules present in the composition of interest. For purposes of thepresent invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote theintracellular destruction of intracellular microbes, or the lysis ofcells infected with such microbes. Another aspect of cellular immunityinvolves an antigen-specific response by helper T-cells. Helper T-cellsact to help stimulate the function, and focus the activity of,nonspecific effector cells against cells displaying peptide antigens inassociation with MHC molecules on their surface. A Acellular immuneresponse@ also refers to the production of cytokines, chemokines andother such molecules produced by activated T-cells and/or other whiteblood cells, including those derived from CD4+ and CD8+ T-cells.

A composition, such as an immunogenic composition, or vaccine thatelicits a cellular immune response may serve to sensitize a vertebratesubject by the presentation of antigen in association with MHC moleculesat the cell surface. The cell-mediated immune response is directed at,or near, cells presenting antigen at their surface. In addition,antigen-specific T-lymphocytes can be generated to allow for the futureprotection of an immunized host.

The ability of a particular antigen or composition to stimulate acell-mediated immunological response may be determined by a number ofassays, such as by lymphoproliferation (lymphocyte activation) assays,CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific forthe antigen in a sensitized subject. Such assays are well known in theart. See, e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doeet al., Eur. J. Immunol. (1994) 24:2369-2376; and the examples below.

Thus, an immunological response as used herein may be one whichstimulates the production of CTLs, and/or the production or activationof helper T-cells. The antigen of interest may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by B-cells; and/or the activation of suppressor T-cellsand/or γδT-cells directed specifically to an antigen or antigens presentin the composition or vaccine of interest. These responses may serve toneutralize infectivity, and/or mediate antibody-complement, or antibodydependent cell cytotoxicity (ADCC) to provide protection to an immunizedhost. Such responses can be determined using standard immunoassays andneutralization assays, well known in the art.

A composition which contains a selected antigen adsorbed to amicroparticle, displays “enhanced immunogenicity” when it possesses agreater capacity to elicit an immune response than the immune responseelicited by an equivalent amount of the antigen when delivered withoutassociation with the microparticle. Thus, a composition may display“enhanced immunogenicity” because the antigen is more stronglyimmunogenic by virtue of adsorption to the microparticle, or because alower dose of antigen is necessary to achieve an immune response in thesubject to which it is administered. Such enhanced immunogenicity can bedetermined by administering the microparticle/antigen composition, andantigen controls to animals and comparing antibody titers against thetwo using standard assays such as radioimmunoassay and ELISAs, wellknown in the art.

The terms “effective amount” or “pharmaceutically effective amount” of amacromolecule/microparticle, as provided herein, refer to a nontoxic butsufficient amount of the macromolecule/microparticle to provide thedesired response, such as an immunological response, and correspondingtherapeutic effect, or in the case of delivery of a therapeutic protein,an amount sufficient to effect treatment of the subject, as definedbelow. As will be pointed out below, the exact amount required will varyfrom subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the condition being treated,and the particular macromolecule of interest, mode of administration,and the like. An appropriate “effective” amount in any individual casemay be determined by one of ordinary skill in the art using routineexperimentation.

By “vertebrate subject” is meant any member of the subphylum cordata,including, without limitation, mammals such as cattle, sheep, pigs,goats, horses, and humans; domestic animals such as dogs and cats; andbirds, including domestic, wild and game birds such as cocks and hensincluding chickens, turkeys and other gallinaceous birds. The term doesnot denote a particular age. Thus, both adult and newborn animals areintended to be covered.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual along with themicroparticle formulation without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of thecomponents of the composition in which it is contained.

By “physiological pH” or a “pH in the physiological range” is meant a pHin the range of approximately 7.2 to 8.0 inclusive, more typically inthe range of approximately 7.2 to 7.6 inclusive.

As used herein, “treatment” refers to any of (i) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen or disorder in question. Treatmentmay be effected prophylactically (prior to infection) or therapeutically(following infection).

General Methods

The present invention is based on the discovery that the PLA and PLGmicroparticles of the present invention efficiently adsorb biologicallyactive macromolecules. Further, these microparticles adsorb a greatervariety of molecules, including charged and/or bulky macromolecules,more readily than microparticles prepared with PVA. Thus themacromolecule/microparticle of the present invention can be used as adelivery system to deliver the biologically active components in orderto treat, prevent and/or diagnose a wide variety of diseases.

The present invention can be used to deliver a wide variety ofmacromolecules including, but not limited to, pharmaceuticals such asantibiotics and antiviral agents, nonsteroidal antiinflammatory drugs,analgesics, vasodilators, cardiovascular drugs, psychotropics,neuroleptics, antidepressants, antiparkinson drugs, beta blockers,calcium channel blockers, bradykinin inhibitors, ACE-inhibitors,vasodilators, prolactin inhibitors, steroids, hormone antagonists,antihistamines, serotonin antagonists, heparin, chemotherapeutic agents,antineoplastics and growth factors, including but not limited to PDGF,EGF, KGF, IGF-1 and IGF-2, FGF, polynucleotides which encode therapeuticor immunogenic proteins, immunogenic proteins and epitopes thereof foruse in vaccines, hormones including peptide hormones such as insulin,proinsulin, growth hormone, GHRH, LHRH, EGF, somatostatin, SNX-111, BNP,insulinotropin, ANP, FSH, LH, PSH and hCG, gonadal steroid hormones(androgens, estrogens and progesterone), thyroid-stimulating hormone,inhibin, cholecystokinin, ACTH, CRF, dynorphins, endorphins, endothelin,fibronectin fragments, galanin, gastrin, insulinotropin, glucagon,GTP-binding protein fragments, guanylin, the leukokinins, magainin,mastoparans, dermaseptin, systemin, neuromedins, neurotensin,pancreastatin, pancreatic polypeptide, substance P, secretin, thymosin,and the like, enzymes, transcription or translation mediators,intermediates in metabolic pathways, immunomodulators, such as any ofthe various cytokines including interleukin-1, interleukin-2,interleukin-3, interleukin-4, and gamma-interferon, antigens, andadjuvants.

In a preferred embodiment the macromolecule is an antigen. A particularadvantage of the present invention is the ability of the microparticleswith adsorbed antigen to generate cell-mediated immune responses in avertebrate subject. The ability of the antigen/microparticles of thepresent invention to elicit a cell-mediated immune response against aselected antigen provides a powerful tool against infection by a widevariety of pathogens. Accordingly, the antigen/microparticles of thepresent invention can be incorporated into vaccine compositions.

Thus, in addition to a conventional antibody response, the system hereindescribed can provide for, e.g., the association of the expressedantigens with class I MHC molecules such that an in vivo cellular immuneresponse to the antigen of interest can be mounted which stimulates theproduction of CTLs to allow for future recognition of the antigen.Furthermore, the methods may elicit an antigen-specific response byhelper T-cells. Accordingly, the methods of the present invention willfind use with any macromolecule for which cellular and/or humoral immuneresponses are desired, preferably antigens derived from viral pathogensthat may induce antibodies, T-cell helper epitopes and T-cell cytotoxicepitopes. Such antigens include, but are not limited to, those encodedby human and animal viruses and can correspond to either structural ornon-structural proteins.

The microparticles of the present invention are particularly useful forimmunization against intracellular viruses which normally elicit poorimmune responses. For example, the present invention will find use forstimulating an immune response against a wide variety of proteins fromthe herpesvirus family, including proteins derived from herpes simplexvirus (HSV) types 1 and 2, such as HSV-1 and HSV-2 glycoproteins gB, gDand gH; antigens derived from varicella zoster virus (VZV), Epstein-Barrvirus (EBV) and cytomegalovirus (CMV) including CMV gB and gH; andantigens derived from other human herpesviruses such as HHV6 and HHV7.(See, e.g. Chee et al., Cytomegaloviruses (J. K. McDougall, ed.,Springer-Verlag 1990) pp. 125-169, for a review of the protein codingcontent of cytomegalovirus; McGeoch et al., J. Gen. Virol. (1988)69:1531-1574, for a discussion of the various HSV-1 encoded proteins;U.S. Pat. No. 5,171,568 for a discussion of HSV-1 and HSV-2 gB and gDproteins and the genes encoding therefor; Baer et al., Nature (1984)310:207-211, for the identification of protein coding sequences in anEBV genome; and Davison and Scott, J. Gen. Virol. (1986) 67:1759-1816,for a review of VZV.)

Antigens from the hepatitis family of viruses, including hepatitis Avirus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the deltahepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus(HGV), can also be conveniently used in the techniques described herein.By way of example, the viral genomic sequence of HCV is known, as aremethods for obtaining the sequence. See, e.g., International PublicationNos. WO 89/04669; WO 90/11089; and WO 90/14436. The HCV genome encodesseveral viral proteins, including E1 (also known as E) and E2 (alsoknown as E2/NSI) and an N-terminal nucleocapsid protein (termed “core”)(see, Houghton et al., Hepatology (1991) 14:381-388, for a discussion ofHCV proteins, including E1 and E2). Each of these proteins, as well asantigenic fragments thereof, will find use in the present compositionand methods.

Similarly, the sequence for the 6-antigen from HDV is known (see, e.g.,U.S. Pat. No. 5,378,814) and this antigen can also be conveniently usedin the present composition and methods. Additionally, antigens derivedfrom HBV, such as the core antigen, the surface antigen, sAg, as well asthe presurface sequences, pre-S 1 and pre-S2 (formerly called pre-S), aswell as combinations of the above, such as sAg/pre-S 1, sAg/pre-S2,sAg/pre-S1/pre-S2, and pre-S1/pre-S2, will find use herein. See, e.g.,AHBV Vaccines—from the laboratory to license: a case study@ in Mackett,M. and Williamson, J. D., Human Vaccines and Vaccination, pp. 159-176,for a discussion of HBV structure; and U.S. Pat. Nos. 4,722,840,5,098,704, 5,324,513, incorporated herein by reference in theirentireties; Beames et al., J. Virol. (1995) 69:6833-6838, Birnbaum etal., J. Virol. (1990) 64:3319-3330; and Zhou et al., J. Virol. (1991)65:5457-5464.

Antigens derived from other viruses will also find use in the claimedcompositions and methods, such as without limitation, proteins frommembers of the families Picornaviridae (e.g., polioviruses, etc.);Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, etc.);Flaviviridae; Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae(e.g., rabies virus, etc.); Filoviridae; Paramyxoviridae (e.g., mumpsvirus, measles virus, respiratory syncytial virus, etc.);Orthomyxoviridae (e.g., influenza virus types A, B and C, etc.);Bunyaviridae; Arenaviridae; Retroviradae (e.g., HTLV-1; HTLV-II; HIV-1(also known as HTLV-III, LAV, ARV, hTLR, etc.)), including but notlimited to antigens from the isolates HIV_(IIIlb), HIV_(SF2), HIV_(LAV),HIV_(LAI), HIV_(MIN)); HIV-1_(CM235), HIV-1_(US4); HIV-2; simianimmunodeficiency virus (SIV) among others. Additionally, antigens mayalso be derived from human papillomavirus (HPV) and the tick-borneencephalitis viruses. See, e.g. Virology, 3rd Edition (W. K. Joklik ed.1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe,eds. 1991), for a description of these and other viruses.

More particularly, the gp120 envelope proteins from any of the above HIVisolates, including members of the various genetic subtypes of HIV, areknown and reported (see, e.g., Myers et al., Los Alamos Database, LosAlamos National Laboratory, Los Alamos, New Mexico (1992); Myers et al.,Human Retroviruses and Aids, 1990, Los Alamos, New Mexico: Los AlamosNational Laboratory; and Modrow et al., J. Virol. (1987) 61:570-578, fora comparison of the envelope sequences of a variety of HIV isolates) andantigens derived from any of these isolates will find use in the presentmethods. Furthermore, the invention is equally applicable to otherimmunogenic proteins derived from any of the various HIV isolates,including any of the various envelope proteins such as gp160 and gp41,gag antigens such as p24gag and p55gag, as well as proteins derived fromthe pol region.

Influenza virus is another example of a virus for which the presentinvention will be particularly useful. Specifically, the envelopeglycoproteins HA and NA of influenza A are of particular interest forgenerating an immune response. Numerous HA subtypes of influenza A havebeen identified (Kawaoka et al., Virology (1990) 179:759-767; Webster etal., “Antigenic variation among type A influenza viruses,” p. 127-168.In: P. Palese and D. W. Kingsbury (ed.), Genetics of influenza viruses.Springer-Verlag, New York). Thus, proteins derived from any of theseisolates can also be used in the compositions and methods describedherein.

The compositions and methods described herein will also find use withnumerous bacterial antigens, such as those derived from organisms thatcause diphtheria, cholera, tuberculosis, tetanus, pertussis, meningitis,and other pathogenic states, including, without limitation, Bordetellapertussis, Neisseria meningitides (A, B, C, Y), Neisseria gonorrhoeae,Helicobacter pylori, and Haemophilus influenza. Hemophilus influenzatype B (HIB), Helicobacter pylori, and combinations thereof. Examples ofantigens from Neisseria meningitides B are disclosed in the followingco-owned patent applications: PCT/US99/09346; PCT “398/01665; PCT1399/00103; and U.S. Provisional Application Ser. Nos. 60/083,758;60/094,869; 60/098,994; 60/103,749; 60/103,794; 60/103,796; and60/121,528. Examples of parasitic antigens include those derived fromorganisms causing malaria and Lyme disease.

It is readily apparent that the subject invention can be used to delivera wide variety of macromolecules and hence to treat, prevent and/ordiagnose a large number of diseases. In an alternative embodiment, themacromolecule/microparticle compositions of the present invention can beused for site-specific targeted delivery. For example, intravenousadministration of the macromolecule/microparticle compositions can beused for targeting the lung, liver, spleen, blood circulation, or bonemarrow.

The adsorption of macromolecules to the surface of the adsorbentmicroparticles occurs via any bonding-interaction mechanism, including,but not limited to, ionic bonding, hydrogen bonding, covalent bonding,Van der Waals bonding, and bonding through hydrophilic/hydrophobicinteractions. Those of ordinary skill in the art may readily selectdetergents appropriate for the type of macromolecule to be adsorbed.

For example, microparticles manufactured in the presence of chargeddetergents, such as anionic or cationic detergents, may yieldmicroparticles with a surface having a net negative or a net positivecharge, which can adsorb a wide variety of molecules. For example,microparticles manufactured with anionic detergents, such as sodiumdodecyl sulfate (SDS), i.e. SDS-PLG microparticles, adsorb positivelycharged antigens, such as proteins. Similarly, microparticlesmanufactured with cationic detergents, such ashexadecyltrimethylammonium bromide (CTAB), i.e. CTAB-PLG microparticles,adsorb negatively charged macromolecules, such as DNA. Where themacromolecules to be adsorbed have regions of positive and negativecharge, either cationic or anionic detergents may be appropriate.

Biodegradable polymers for manufacturing microparticles for use with thepresent invention are readily commercially available from, e.g.,Boehringer Ingelheim, Germany and Birmingham Polymers, Inc., Birmingham,Ala. For example, useful polymers for forming the microparticles hereininclude those derived from polyhydroxybutyric acid; polycaprolactone;polyorthoester; polyanhydride; as well as a poly(a-hydroxy acid), suchas poly(L-lactide), poly(D,L-lactide) (both known as APLA” herein),poly(hydoxybutyrate), copolymers of D,L-lactide and glycolide, such aspoly(D,L-lactide-co-glycolide) (designated as “PLG@ or “PLGA” herein) ora copolymer of D,L-lactide and caprolactone. Particularly preferredpolymers for use herein are PLA and PLG polymers. These polymers areavailable in a variety of molecular weights, and the appropriatemolecular weight for a given use is readily determined by one of skillin the art. Thus, e.g., for PLA, a suitable molecular weight will be onthe order of about 2000 to 5000. For PLG, suitable molecular weightswill generally range from about 10,000 to about 200,000, preferablyabout 15,000 to about 150,000, and most preferably about 50,000 to about100,000.

If a copolymer such as PLG is used to form the microparticles, a varietyof lactide:glycolide ratios will find use herein and the ratio islargely a matter of choice, depending in part on the coadministeredmacromolecule and the rate of degradation desired. For example, a 50:50PLG polymer, containing 50% D,L-lactide and 50% glycolide, will providea fast resorbing copolymer while 75:25 PLG degrades more slowly, and85:15 and 90:10, even more slowly, due to the increased lactidecomponent. It is readily apparent that a suitable ratio oflactide:glycolide is easily determined by one of skill in the art basedon the nature of the antigen and disorder in question. Moreover,mixtures of microparticles with varying lactide:glycolide ratios willfind use herein in order to achieve the desired release kinetics for agiven macromolecule and to provide for both a primary and secondaryimmune response. Degradation rate of the microparticles of the presentinvention can also be controlled by such factors as polymer molecularweight and polymer crystallinity. PLG copolymers with varyinglactide:glycolide ratios and molecular weights are readily availablecommercially from a number of sources including from BoehringerIngelheim, Germany and Birmingham Polymers, Inc., Birmingham, Ala. Thesepolymers can also be synthesized by simple polycondensation of thelactic acid component using techniques well known in the art, such asdescribed in Tabata et al., J. Biomed. Mater. Res. (1988) 22:837-858.

The macromolecule/microparticles are prepared using any of severalmethods well known in the art. For example, double emulsion/solventevaporation techniques, such as those described in U.S. Pat. No.3,523,907 and Ogawa et al., Chem. Pharm. Bull. (1988) 36:1095-1103, canbe used herein to make the microparticles. These techniques involve theformation of a primary emulsion consisting of droplets of polymersolution, which is subsequently mixed with a continuous aqueous phasecontaining a particle stabilizer/surfactant.

Alternatively, a water-in-oil-in-water (w/o/w) solvent evaporationsystem can be used to form the microparticles, as described by O=Haganet al., Vaccine (1993) 11:965-969 and Jeffery et al., Pharm. Res. (1993)10:362. In this technique, the particular polymer is combined with anorganic solvent, such as ethyl acetate, dimethylchloride (also calledmethylene chloride and dichloromethane), acetonitrile, acetone,chloroform, and the like. The polymer will be provided in about a 1-30%,preferably about a 2-15%, more preferably about a 3-10% and mostpreferably, about a 4% solution, in organic solvent. The polymersolution is emulsified using e.g., an homogenizer. The emulsion is thenoptionally combined with a larger volume of an aqueous solution of anemulsion stabilizer such as polyvinyl alcohol (PVA), polyvinylpyrrolidone, and a cationic, anionic, or nonionic detergent. Theemulsion may be combined with more than one emulsion stabilizer and/ordetergent, e.g., a combination of PVA and a detergent. Certainmacromolecules may adsorb more readily to microparticles having acombination of stabilizers and/or detergents. Where an emulsionstabilizer is used, it is typically provided in about a 2-15% solution,more typically about a 4-10% solution. Generally, a weight to weightdetergent to polymer ratio in the range of from about 0.00001:1 to about0.1:1 will be used, more preferably from about 0.0001:1 to about 0.01:1,more preferably from about 0.001:1 to about 0.01:1, and even morepreferably from about 0.005:1 to about 0.01:1. The mixture is thenhomogenized to produce a stable w/o/w double emulsion. Organic solventsare then evaporated.

The formulation parameters can be manipulated to allow the preparationof small microparticles on the order of 0.05 μm (50 nm) to largermicroparticles 50 μm or even larger. See, e.g., Jeffery et al., Pharm.Res. (1993) 10:362-368; McGee et al., J. Microencap. (1996). Forexample, reduced agitation results in larger microparticles, as does anincrease in internal phase volume. Small particles are produced by lowaqueous phase volumes with high concentrations of emulsion stabilizers.

Microparticles can also be formed using spray-drying and coacervation asdescribed in, e.g., Thomasin et al., J. Controlled Release (1996)41:131; U.S. Pat. No. 2,800,457; Masters, K. (1976) Spray Drying 2nd Ed.Wiley, New York; air-suspension coating techniques, such as pan coatingand Wurster coating, as described by Hall et al., (1980) The AWursterProcess@ in Controlled Release Technologies: Methods, Theory, andApplications (A. F. Kydonieus, ed.), Vol. 2, pp. 133-154 CRC Press, BocaRaton, Fla. and Deasy, P. B., Crit. Rev. Ther. Drug Carrier Syst. (1988)S(2):99-139; and ionic gelation as described by, e.g., Lim et al.,Science (1980) 210:908-910.

Particle size can be determined by, e.g., laser light scattering, usingfor example, a spectrometer incorporating a helium-neon laser.Generally, particle size is determined at room temperature and involvesmultiple analyses of the sample in question (e.g., 5-10 times) to yieldan average value for the particle diameter. Particle size is alsoreadily determined using scanning electron microscopy (SEM).

Following preparation, microparticles can be stored as is orfreeze-dried for future use. In order to adsorb macromolecules to themicroparticles, the microparticle preparation is simply mixed with themacromolecule of interest and the resulting formulation can again belyophilized prior to use. Generally, macromolecules are added to themicroparticles to yield microparticles with adsorbed macromoleculeshaving a weight to weight ratio of from about 0.0001:1 to 0.25:1macromolecules to microparticles, preferably, 0.001:1 to 0.1, morepreferably 0.01 to 0.05. Macromolecule content of the microparticles canbe determined using standard techniques.

The microparticles of the present invention may have macromoleculesentrapped or encapsulated within them, as well as having macromoleculesadsorbed thereon. Thus, for example, one of skill in the art may preparein accordance with the invention microparticles having encapsulatedadjuvants with proteins adsorbed thereon, or microparticles havingencapsulated proteins with adjuvants adsorbed thereon.

Once the macromolecule adsorbed microparticles are produced, they areformulated into pharmaceutical compositions or vaccines, to treat,prevent and/or diagnose a wide variety of disorders, as described above.The compositions will generally include one or more Apharmaceuticallyacceptable excipients or vehicles@ such as water, saline, glycerol,polyethylene-glycol, hyaluronic acid, ethanol, etc. Additionally,auxiliary substances, such as wetting or emulsifying agents, biologicalbuffering substances, and the like, may be present in such vehicles. Abiological buffer can be virtually any solution which ispharmacologically acceptable and which provides the formulation with thedesired pH, i.e., a pH in the physiological range. Examples of buffersolutions include saline, phosphate buffered saline, Tris bufferedsaline, Hank's buffered saline, and the like.

Adjuvants may be used to enhance the effectiveness of the pharmaceuticalcompositions. The adjuvants may be administered concurrently with themicroparticles of the present invention, e.g., in the same compositionor in separate compositions. Alternatively, an adjuvant may beadministered prior or subsequent to the microparticle compositions ofthe present invention. In another embodiment, the adjuvant, such as animmunological adjuvant, may be encapsulated in the microparticle.Adjuvants, just as any macromolecules, may be encapsulated within themicroparticles using any of the several methods known in the art. See,e.g., U.S. Pat. No. 3,523,907; Ogawa et al., Chem Pharm. Bull (1988)36:1095-1103; O=Hagan et al., Vaccine (1993) 11:965-969 and Jefferey etal., Pharm. Res. (1993) 10:362. Alternatively, adjuvants may be adsorbedon the microparticle as described above for any macromolecule.

Immunological adjuvants include, but are not limited to: (1) aluminumsalts (alum), such as aluminum hydroxide, aluminum phosphate, aluminumsulfate, etc.; (2) oil-in-water emulsion formulations (with or withoutother specific immunostimulating agents such as muramyl peptides (seebelow) or bacterial cell wall components), such as for example (a) MF59(International Publication No. WO 90/14837), containing 5% Squalene,0.5% Tween 80, and 0.5% Span 85 (optionally containing various amountsof MTP-PE (see below), although not required) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below)either microfluidized into a submicron emulsion or vortexed to generatea larger particle size emulsion, and (c) RibiJ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (DetoxJ) (for a furtherdiscussion of suitable submicron oil-in-water emulsions for use herein,see commonly owned, patent application Ser. No. 09/015,736, filed onJan. 29, 1998); (3) saponin adjuvants, such as QS21 (e.g., StimulonJ(Cambridge Bioscience, Worcester, Mass.)) may be used or particlegenerated therefrom such as ISCOMs (immunostimulating complexes); (4)Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA);(5) cytokines, such as interleukins (1L-1, IL-2, etc.), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.;(6) detoxified mutants of a bacterial ADP-ribosylating toxin such as acholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labiletoxin (LT), particularly LT-K63 (where lysine is substituted for thewild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S 109(where serine is substituted for the wild-type amino acid at position109), and PT-K9/G129 (where lysine is substituted for the wild-typeamino acid at position 9 and glycine substituted at position 129) (see,e.g., International Publication Nos. WO93/13202 and WO92/19265); (7) CpGoligonucleotides and other immunostimulating sequences (ISSs); and (8)other substances that act as immunostimulating agents to enhance theeffectiveness of the composition. Alum and MF59 are preferred.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

For additional examples of adjuvants, see Vaccine Design, The Subunitand the Adjuvant Approach, Powell, M. F. and Newman, M. J, eds., PlenumPress, 1995).

The compositions will comprise a Atherapeutically effective amount@ ofthe macromolecule of interest. That is, an amount ofmacromolecule/microparticle will be included in the compositions whichwill cause the subject to produce a sufficient response, in order toprevent, reduce, eliminate or diagnose symptoms. The exact amountnecessary will vary, depending on the subject being treated; the age andgeneral condition of the subject to be treated; the severity of thecondition being treated; in the case of an immunological response, thecapacity of the subject's immune system to synthesize antibodies; thedegree of protection desired and the particular antigen selected and itsmode of administration, among other factors. An appropriate effectiveamount can be readily determined by one of skill in the art. Thus, aAtherapeutically effective amount@ will fall in a relatively broad rangethat can be determined through routine trials. For example, for purposesof the present invention, where the macromolecule is a polynucleotide,an effective dose will typically range from about 1 ng to about 1 mg,more preferably from about 10 ng to about 1 μg, and most preferablyabout 50 ng to about 500 ng of the macromolecule delivered per dose;where the macromolecule is an antigen, an effective dose will typicallyrange from about 1 μg to about 100 mg, more preferably from about 10 μgto about 1 mg, and most preferably about 50 μg to about 500 μg of themacromolecule delivered per dose.

Once formulated, the compositions of the invention can be administeredparenterally, e.g., by injection. The compositions can be injectedeither subcutaneously, intraperitoneally, intravenously orintramuscularly. Other modes of administration include nasal, oral andpulmonary administration, suppositories, and transdermal ortranscutaneous applications. Dosage treatment may be a single doseschedule or a multiple dose schedule. A multiple dose schedule is one inwhich a primary course of administration may be with 1-10 separatedoses, followed by other doses given at subsequent time intervals,chosen to maintain and/or reinforce the therapeutic response, forexample at 1-4 months for a second dose, and if needed, a subsequentdose(s) after several months. The dosage regimen will also, at least inpart, be determined by the need of the subject and be dependent on thejudgment of the practitioner.

Furthermore, if prevention of disease is desired, the macromolecules invaccines, are generally administered prior to primary infection with thepathogen of interest. If treatment is desired, e.g., the reduction ofsymptoms or recurrences, the macromolecules are generally administeredsubsequent to primary infection.

C. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXAMPLE 1 Preparation of Blank Microparticles Using PVA as an EmulsionStabilizer

Blank microparticles (e.g., without adsorbed or entrappedmacromolecules) were made using polyvinyl alcohol (PVA) as follows.Solutions used:

-   -   (1) 6% RG 504 PLG (Boehringer Ingelheim) in dichloromethane.    -   (2) 10% polyvinyl alcohol (PVA) (ICN) in water.

In particular, the microparticles were made by combining 10 ml ofpolymer solution with 1.0 ml of distilled water and homogenizing for 3minutes using an Omni benchtop homogenizer with a 10 mm probe at 10K rpmto form a water/oil (w/o) emulsion. The w/o emulsion was added to 40 mlof the 10% PVA solution, and homogenized for 3 minutes, to form awater/oil/water (w/o/w) emulsion. The w/o/w emulsion was left stirringovernight for solvent evaporation, forming microparticles. The formedmicroparticles were washed with water by centrifugation 4 times, andlyophilized. The microparticles were then sized in a Malvern Mastersizer for future use.

EXAMPLE 2 Preparation of Blank Microparticles Using CTAB

Blank microparticles were produced using CTAB as follows. Solutionsused:

-   -   (1) 4% RG 504 PLG (Boehringer Ingelheim) in dimethyl chloride.    -   (2) 0.5% CTAB (Sigma Chemical Co., St. Louis, Mo.) in water.

In particular, the microparticles were made by combining 12.5 ml ofpolymer solution with 1.25 ml of distilled water and homogenizing for 3minutes using an Omni benchtop homogenizer with a 10 mm probe at 10K rpmto form a w/o emulsion. The w/o emulsion was added to 50 ml of the 0.5%CTAB solution and homogenized for 3 minutes to form a w/o/w emulsion.The w/o/w emulsion was left stirring overnight for solvent evaporation,forming microparticles. The formed microparticles were then filteredthrough a 38μ mesh, washed with water by centrifugation 4 times, andlyophilized. The microparticles were then sized in a Malvern Mastersizer for future use.

EXAMPLE 3 Preparation of Blank Microparticles Using SDS

Blank microparticles were produced using SDS as follows. Solutions used:

-   -   (1) 6% RG 504 PLG (Boehringer Ingelheim) in dimethyl chloride.    -   (2) 1% SDS (Sigma Chemical Co., St. Louis, Mo.) in water.

In particular, the microparticles were made by combining 12.5 ml ofpolymer solution with 50 ml of the SDS solution and homogenizing for 3minutes using an Omni benchtop homogenizer with a 10 mm probe at 10Krpm. The emulsion was left stirring overnight for solvent evaporation.The formed microparticles were filtered through a 38μ mesh, washed withwater by centrifugation 4 times, and lyophilized for future use. Themicroparticles were then sized in a Malvern Master sizer for future use.

EXAMPLE 4 Adsorption of Protein to Blank Microparticles

Protein was adsorbed to microparticles as follows.

-   -   A. 1% and 3% theoretical load of p55gag

In order to achieve 1% and 3% theoretical loads, 50 mg of thelyophilized blank SDS/PLG microparticles produced as in Example 3 wereplaced in a Nalgene centrifuge tube and 10 ml of 25 mM Borate buffer, pH9, with 6M urea containing p55gag protein (Chiron Corporation, Berkeley,Calif.) was added: (a) for 1% theoretical load 10 ml of a 501 g/ml p55gag solution was used; and (b) for 3% theoretical load 10 ml of a 150μg/ml p55 gag solution was used. The mixture was incubated with rockingovernight at room temperature. The next day, the microparticles werecentrifuged and the supernatant assayed by a bicinchoninic assay (BCA;Pierce, Rockford, Ill.), for gag concentration to determine the amountadsorbed. The microparticles were washed twice with 10 ml Borate/6M ureabuffer and twice with 30 ml water, and lyophilized for future use.

B. 1% theoretical load of HCV Core Antigen

In order to achieve 1% theoretical load, 50 mg of the lyophilized blankSDS/PLG microparticles were placed in a Nalgene centrifuge tube and 10ml of 30 mM citrate buffer, pH 6.5, with 6M urea containing monomericHCV core protein (10 ml of a 50 μg/ml HCV core protein solution; ChironCorporation, Berkeley, Calif.) was added. The mixture was incubated withrocking overnight at room temperature. The next day, the microparticleswere centrifuged and the supernatant assayed by a bicinchoninic assay(BCA; Pierce, Rockford, IL), for HCV concentration to determine theamount adsorbed. The microparticles were washed twice with 30 mlcitrate/6M urea buffer and twice with 30 ml water, and lyophilized forfuture use.

EXAMPLE 5 Adsorption Efficiency of Microparticles

The lyophilized microparticles with adsorbed protein from Example 4 wereanalyzed for total adsorbed protein using base hydrolysis as follows. 10mg of the lyophilized adsorbed particles were hydrolyzed for four hoursin 2 ml 0.2N NaOH with 5% SDS, neutralized, and diluted 1:10 andanalyzed for protein content using the MicroBCA protein assay (Pierce,Rockford, Ill.). As shown in Table 1, microparticles with modifiedsurfaces prepared with detergents like CTAB and SDS, both adsorbedprotein more efficiently than microparticles made using solely PVA.TABLE 1 Microparticle Targeted Load Actual Load Type Protein (% w/w) (%w/w) PVA-PLG p55gag 3% 0.38% CTAB-PLG p55gag 3% 1.58% SDS-PLG p55gag 3%1.36% PVA-PLG p55gag 1% 0.18% SDS-PLG p55gag 0.5%   0.45% SDS-PLG p55gag1% 0.72% SDS-PLG p55gag 1% 0.79% PVA-PLG HCV Core 4%  0.3% SDS-PLG HCVCore 1%  0.7%

EXAMPLE 6 A. Immunogenicity of gag-Adsorbed Microparticles

The gag-adsorbed microparticles, produced using PVA or SDS, as describedin Example 4, as well as p5sgag alone, without associated microparticles(as a negative control) and vaccinia gag-pol controls (as a positivecontrol) were administered intramuscularly to mice. The animals wereboosted at 7 and 14 days. The total dose administered is indicated inTables 2 and 3. Spleens were collected two weeks following the lastimmunization and CTL activity assayed as described in Doe et al., Proc.Natl. Acad. Sci. (1996) 93:8578-8583.

The lymphocyte cultures were prepared as follows. Spleen cells (sc) fromimmunized mice were cultured in 24-well dishes at 5×10⁶ cells per well.Of those cells, 1×10⁶ were sensitized with synthetic epitopic peptidesform HIV-1_(SF2) proteins at a concentration of 10 μM for 1 hour at37EC, washed, and cocultured with the remaining 4×10⁶ untreated sc in 2ml of culture medium [50% RPMI 1640 and 50% alpha-MEM (GIBCO)]supplemented with heat-inactivated fetal calf serum, 5×10⁻⁵ M2-mercaptoethanol, antibiotics, and 5% interleukin 2 (Rat T-Stim,Collaborative Biomedical Products, Bedford, Mass.). Cells were fed with1 ml of fresh culture medium on days 3 and 5, and cytotoxicity wasassayed on day 6.

The cytotoxic cell assay was conducted as follows. SvBALB (H-2^(d))(SvB) and MC57 (H-2^(b)) target cells used in the ⁵¹Cr release assaysexpress class I but not class II MHC molecules. Approximately 1×10⁶target cells were incubated in 200 μl of medium containing 50 μCi (1Ci=37 Gbq) of ⁵¹Cr and synthetic HIV-1 peptides (1 mM) for 60 min andwashed three times. Effector (E) cells were cultured with 5×10³ target(T) cells at various E/T ratios in 200 μl of culture medium in 96-wellround-bottom tissue culture plates for 4 hours. The average cpm fromduplicate wells was used to calculate percent specific ⁵¹Cr release.

As shown in Tables 2 and 3, the SDS-PLG/p55 microparticles had activitycomparable to the vaccinia control and was more active than thePVA-PLG/p55 microparticles and the p55gag protein formulation.Specifically, as shown in Table 2, p55gag protein were inactive atconcentrations of 10 μg, 25 μg and 50 μg. Further, as shown in Table 3,the SDS-PLG/p55 formulations were more active than the PVA-PLG/p55 andp55gag protein formulations, indicating that proteins were adsorbed moreefficiently to the microparticles in the SDS-PLG/p55 formulations ascompared to the PVA-PLG/p55 and p55gag protein formulations. TABLE 2PERCENT SPECIFIC LYSIS OF TARGETS Antigen Adjuvant Target SvB MC57 (Adj.Dose) Ratio SvB^(a) P7g+^(b) p7G−^(c) p55gag protein 60 15 12 4 (10 μg)15 11 8 3 4 7 6 3 % Spon Release 12 10 13 p55gag protein 63 10 18 2 (25μg) 16 7 6 −1 4 4 1 −3 % Spon Release 12 10 13 p55gag protein 60 28 22 5(50 μg) 15 13 12 2 4 9 3 3 % Spon Release 12 10 13 p55gag protein 60 850 0 (10 μg)PLG/ 15 5 21 −3 SDS 0.6% 11.6 mg 4 4 7 −1 % Spon Release 1210 13 Vv gag/pol 60 9 65 1 (vaccinia virus 15 4 38 1 encoding gag) 4 118 3 % Spon Release 12 10 10 13^(a)SvB cell line without peptide pulsing^(b)SvB cell line pulsed with p7g peptide^(c)MC57 cell line pulsed with p7g peptide

TABLE 3 PERCENT SPECIFIC LYSIS OF TARGETS E:T MC57 + SVB + EffectorRatio MC57^(a) gag b^(b) gag b^(c) PVA-PLG/p55 60:1 8 15 11 10 μg 12:1 310 2 2.4:1  >1 5 2 SDS-PLG/p55 60:1 6 35 4 10 μg 12:1 3 12 >1 2.4:1  >13 2 p55gag protein 60:1 7 15 1 10 μg 12:1 2 6 1 2.4:1  >1 1 >1 Vacciniagag 60:1 >1 37 >1 12:1 >1 19 >1 2.4:1  1 9 >1^(a)MC57 cell line without pulsing with peptide^(b)MC57 cell line pulsed with gag b peptide^(c)SVB cell line pulsed with gag b peptide

EXAMPLE 7 Preparation of pCMVgp120 DNA-Adsorbed Microparticles withModified Surfaces

Microparticles with adsorbed plasmid DNA encoding gp120 were prepared asfollows. 20 mg of blank microparticles, prepared as described inExamples 1 and 2, were incubated with increasing concentrations ofpCMVgp120 DNA in a 1.0 ml volume for 3 hours at 4° C. Followingincubation, the microparticles were centrifuged, washed twice withTris-EDTA buffer and freeze-dried overnight. The microparticles werehydrolyzed as described in Example 5 and analyzed for the amount ofadsorbed DNA at A₂₆₀ nm.

Table 4 illustrates the loading efficiency of PLG-PVA and PLG-CTABmicroparticles. As indicated in the table, the PLG-CTAB microparticlesadsorb more efficiently than the corresponding PLG-PVA particles. TABLE4 Microparticle Theoretical Actual Load Loading Type Load (% w/w) (%w/w) Efficiency (% w/w) PLG-PVA 1 0.44 44 PLG-CTAB 1 0.84 88 PLG-PVA 20.38 19 PLG-CTAB 2 1.23 62 PLG-PVA 3 0.33 11 PLG-CTAB 3 1.82 61 PLG-PVA4 0.48 12 PLG-CTAB 4 2.36 59

EXAMPLE 8 HCV-E2 Adsorption

Microparticles were prepared using PVA, and several differentdetergents, as described in the previous examples. E2 protein fromHepatitus C Virus (HCV) was adsorbed on the surface of themicroparticles as follows: 0.2 mg/ml E2 was added to 20 mg of themicroparticles in PBS to form a solution at 0.5% w/w E2/PLG in a totalvolume of 0.5 ml. The solutions were incubated for 1.5 hours at 37EC,then centrifuged. The supernatants were collected and then measured forprotein content by microBCA. The results are shown in Table 5. Theresults confirm the superior adsorption of macromolecules by themicroparticles of the present invention. TABLE 5 % bound % totalMicroparticle Type Protein (w/w E2/PLG) E2 bound PVA-PLG E2 0.00 0.00CTAB-PLG E2 0.43 96.00 SDS-PLG E2 0.14 31.00 NaOleate-PLG E2 0.36 81.00Pluronic P84-PLG E2 0.00 0.00 Pluronic L121-PLG E2 0.00 0.00

EXAMPLE 9 Adsorption of gp120 Protein

Microparticles were prepared using PVA as described in the previousexamples. Microparticles were also prepared using NaOleate, an anionicdetergent, as follows: a w/o/w emulsion was prepared with 1.67 ml of 30mM NaCltrate at pH6 as the internal water phase, 16.7 ml of 6% polymerRG 505 PLG (Boehringer Ingelheim) in dichloromethane as the solvent (oilphase), and 66.8 ml of 0.4% NaOleate as the external aqueous phase.These microparticles appear in Table 6 below as “NaOleate-PLG (w/o/w).”Additionally, microparticles were prepared using NaOleate in an oil inwater formulation, and these microparticles appear in Table 6 below as“NaOleate-PLG (o/w).” gp120 protein was adsorbed on the surface of theprepared microparticles as follows: 0.388 mg/ml of protein was added toabout 20 mg of the microparticles in PBS to form a solution at about1.4% w/w gp120/PLG in a total volume of 0.8 ml. The solutions wereincubated for 1.5 hours at 37EC, then centrifuged. The supernatants werecollected and then measured for protein content by microBCA. The resultsare shown in Table 6. The results confirm the superior adsorption ofmacromolecules by the microparticles of the present invention. TABLE 6 %bound % total Microparticle Type protein (w/w gp120/PLG) E2 boundPVA-PLG gp120 0.01 0.00 PVA-PLG gp120 0.09 3.00 NaOleate-PLG (w/o/w)gp120 1.33 96.00 NaOleate-PLG (w/o/w) gp120 1.24 95.00 NaOleate-PLG(o/w) gp120 0.41 31.00 NaOleate-PLG (o/w) gp120 0.27 20.00 NaOleate-PLG(o/w) gp120 0.36 28.00 NaOleate-PLG (o/w) gp120 0.27 22.00 NaOleate-PLG(o/w) gp120 0.34 26.00 NaOleate-PLG (o/w) gp120 0.31 24.00 NaOleate-PLG(o/w) gp120 −0.01 −1.00 NaOleate-PLG (o/w) gp120 −0.09 −7.00

EXAMPLE 10 Adsorption of Listeriolysin Protein

Microparticles were prepared using PVA and CTAB, as described in theprevious examples. Listeriolysin protein (LLO) from Listeriamonocytogenes was adsorbed on the surface of the microparticles asfollows: 1.0 mg/ml LLO was added to 100 mg of the microparticles in PBSto form a solution at 1% w/w LLO/PLG in a total volume of 5 ml. Thesolutions were incubated for 1.5 hours at 37EC, then centrifuged. Thesupernatants were collected and then measured for protein content bymicroBCA. The results are shown in Table 7. The results confirm thesuperior adsorption of macromolecules by the microparticles of thepresent invention. TABLE 7 Microparticle Targeted Actual Loading TypeProtein Load (% w/w) Load (% w/w) Efficiency PVA-PLG LLO 0.10 0.10 10.0PVA-PLG LLO 0.25 0.08 32.0 PVA-PLG LLO 0.50 0.12 24.0 PVA-PLG LLO 1.000.18 18.0 CTAB-PLG LLO 0.10 0.06 60.0 CTAB-PLG LLO 0.25 0.19 76.0CTAB-PLG LLO 0.50 0.34 68.0 CTAB-PLG LLO 1.00 0.71 71.0

EXAMPLE 11 Effect of Aluminum Salt as an Adjuvant

p55 gag DNA-adsorbed PLG microparticles were prepared as describedabove, using CTAB. The microparticles were injected intramuscularly inmice at two concentrations, and, as a control, DNA alone was injected atthe same two concentrations. Additionally, in one trial, 50 Φg aluminumphosphate was added to the injected CTAB composition. Each formulationwas injected into ten mice. The mice were boosted after 28 days. Twoweeks after the second immunization, serum was collected and thegeometric mean titer (GMT) of each serum was measured, along with itsstandard error (SE). The results are summarized in Table 8, presented asboth linear and log values. Each number is the average of the resultsobtained from the ten mice. TABLE 8 log log Formulation GMT SE GMT SEDNA-CTAB 1 Φg 19546 5983 4.28 0.11 DNA-CTAB 10 Φg 54487 5510 4.73 0.04DNA-CTAB 1 Φg + 49765 10034 4.69 0.1 ALUM 50 Φg DNA alone 1 Φg 10.6 2.71.01 0.07 DNA alone 10 Φg 230 395 2.15 0.3

In order to compare these results statistically, P-values were generatedfor DNA-CTAB vs. DNA-CTAB+ALUM (P-value=0.0017); DNA-CTAB+ALUM vs. DNAalone (P-value<0.0001); and DNA-CTAB (10 Φg) vs. DNA alone (10 Φg)(P-value<0.0001). These P-values confirm the statistical significance ofthe values in Table 8.

EXAMPLE 12 Measurement of Zeta Potentials

Measurement of zeta potentials was carried out on a DELSA 440 SXzetasizer from Coulter Corp., Miami, Fla. 33116. The system iscalibrated using mobility standards from Coulter (EMP SL7, an aqueoussuspension of polystyrene latex beads). Following rinsing of the samplecell with sterile water, samples are added to the sample cell. Thecounter is then set to zero by aligning the beam to its lowest value.The current is set at 0.7 mA for the reference and 20 V for the sample.Detector levels from all four beams are checked, then the sample is runby selecting “run” from the software, and frequency measurements areread. The beams should be 20 Hz apart. The mean zeta potential for eachsample is then read.

Measurements for several microparticle formulations of the presentinvention were read, and the results are shown in Table 9. As theresults indicate, adsorbance of macromolecules to the microparticles'surfaces alters the zeta potentials of the microparticles. TABLE 9Microparticle Adherent Zeta Potential Type macromolecule (mV) PLG-PVAnone −26 ∀ 8 PLG-CTAB none +83 ∀ 22 PLG-CTAB p55 DNA +35 ∀ 14 PLG-SDSnone −44 ∀ 26 PLG-SDS p55 protein −32 ∀ 18 PLG-Oleate none −64 ∀ 24PLG-Oleate gp120 protein −48 ∀ 14

EXAMPLE 13 Microparticles with Encapsulated and Adsorbed Macromolecules

(A). PLG microparticles were prepared using RG 505 PLG and PVA, andencapsulating the adjuvant LTK63. 100 mg of the microparticles wasincubated with 5 ml PBS containing 400 Φg/ml p24gag protein. The mixturewas then incubated with rocking at room temperature overnight, washed bycentrifugation with 20 ml PBS twice and with water once, thenlyophilized. Following base hydrolysis and neutralization, the %adsorbed protein and % encapsulated adjuvant were measured; the resultsappear in Table 10.

(B). PLG microparticles were prepared using SDS and RG 505 PLG, andencapsulating adjuvant CpG oligonucleotides as follows: 5 ml of 6% RG505polymer in DCM was emulsified with 0.5 ml of 5 mg/ml CpG in 50 mMTris/EDTA, forming a w/o emulsion. The w/o emulsion was added to 20 mlof 1% SDS and then emulsified, forming a w/o/w emulsion. Microparticleswere formed by solvent evaporation overnight, then washed, centrifuged,and lyophilized. 10 mg of the CpG-encapsulated microparticles wasdissolved in 1 ml DCM. 0.5 ml water was added to extract theoligonucleotides, and the mixture was then centrifuged and the aqueouslayer was injected on a size exclusion column with PBS as the mobilephase. 10 mg of placebo microparticles was mixed with 100 Φg CpGoligonucleotides and extracted as above with DCM and run on the columnas a standard. The amount of CpG oligonucleotides present in theentrapped particles was calculated against the standard.

p55gag was adsorbed on the CpG-encapsulated microparticles as follows:50 mg of the lyophilized CpG-encapsulated microparticles was incubatedovernight with 5 ml 25 mM Borate with 6M Urea (pH 9) containing 140 Φgp55gag protein. The mixture was incubated with rocking overnight at roomtemperature, washed with 20 ml Borate buffer/6M Urea twice, and 20 mlwater twice, then lyophilized.

10 mg of the CpG-encapsulated/p55gag adsorbed microparticles was basehydrolyzed, and measurements were taken of the % entrapped and %adsorbed macromolecules. The targeted load was 1.0%, except as otherwiseindicated. The results appear in Table 10. TABLE 10 Microparticle Type %encapsulated (w/w) % adsorbed (w/w) (A). PLG-PVA 0.46  1.2* LTK63encapsulated p24gag adsorbed (B). PLG-SDS 0.41 1.0 CpG encapsulatedp55gag adsorbed*targeted load = 2.0%

EXAMPLE 14 Microparticles with Two Adsorbed Macromolecules

(A). According to the present invention, two or more macromolecules maybe administered in a composition comprising microparticles which haveadsorbed both macromolecules, or may be administered in a compositioncomprising two or more distinct microparticles, each having adsorbed asingle macromolecule. For example, microparticles were preparedadsorbing both E2 polypeptide and adjuvant CpG oligonucleotides asfollows: Blank PLG-CTAB were prepared as previously described. 20 mg ofthe lyophilized microparticles were incubated for 4 hours with 1 ml of200 Φg/ml E2 in saline. The mixture was rocked at room temperature for 4hours, washed with 20 ml of normal saline water twice by centrifugationat 10,000 G, and the pellet was resuspended in 1 ml of a CpG solution inTE buffer containing 200 Φg/ml CpG for 4 hours at room temperature. Thefinal suspension was washed twice with TE buffer by centrifugation, andthen lyophilized. 10 mg of the microparticles with adsorbed CpG and E2was base hydrolyzed and protein concentration was determined by BCA, andthe residual amount of CpG in the supernatant was assayed by HPLC tomeasure the amount of CpG adsorbed on the microparticles. The resultsappear in Table 11, demonstrating positive adsorption for bothmacromolecules.

(B). Microparticles were prepared according to the invention. A portionwere used to adsorb E2 polypeptide, while another portion was used toadsorb adjuvant CpG olignucleotides. Blank PLG-CTAB were prepared aspreviously described. 20 mg of the lyophilized microparticles wereincubated for 4 hours with 1 ml of 200 Φg/ml E2 in saline. The mixturewas rocked at room temperature for 4 hours, washed with 20 ml of normalsaline water twice by centrifugation at 10,000 G, then lyophilized.Separately, 20 mg of the lyophilized microparticles were incubated for 4hours with 1 ml of 200 Φg/ml CpG in TE buffer. The mixture was rocked atroom temperature for 4 hours, washed with 20 ml of TE buffer twice bycentrifugation at 10,000 G, then lyophilized. Results of measurements ofthe percent adsorbed macromolecules appears in Table 11. TABLE 11Microparticle % adsorbed E2 % adsorbed CpG Type (w/w)* (w/w)* (A).PLG-SDS 0.71 0.32 E2 adsorbed CpG adsorbed (B). PLG-SDS 0.64 n/a E2adsorbed (B). PLG-SDS n/a 0.81 CpG adsorbed*targeted load = 1.0%

EXAMPLE 15 Microparticles Formed Using Combination of Detergent and PVA

The following procedure was used to form microparticles comprising twosurfactants: PVA and a detergent: 10 ml of 5% PLG polymer and 0.2% ofthe detergent DOTAP in DCM were emulsified at 12,000 rpm for 3 minuteswith 1.0 ml distilled water to form the primary w/o emulsion. The w/oemulsion was added to 40 ml of 0.8% PVA and emulsified for 3 minutes toform the second w/o/w emulsion, which was stirred overnight to evaporatethe solvent, and microparticles were formed. The microparticles werewashed twice in distilled water and lyophilized. The microparticles arethen ready for adsorption of macromolecules in accordance with thepresent invention.

The same procedure was employed to form microparticles comprising acombination of PVA and the detergent DDA.

EXAMPLE 16

Immunogenicity of Microparticles With Adsorbed 1S5 DNA

Microparticles were formed as in the previous examples using thedetergents CTAB or DDA. p55 DNA was adsorbed to the microparticles andimmunogenicity was assessed using the procedures described in in theprevious examples. The results are summarized in Table 12 below. TABLE12 PERCENT SPECIFIC LYSIS OF TARGETS Effector E:T Ratio Sv/B P7g^(a)PLG-CTAB/ 60:1 71 p55 DNA 15:1 55 1 μg  4:1 31 PLG-DDA/ 60:1 70 p55 15:154 1 μg  4:1 17 p55 DNA alone 60:1 3 1 μg 15:1 1  4:1 0 Vaccinia gag60:1 64 2 × 10⁷ pfu 15:1 35  4:1 11^(a)SVB cell line pulsed with gag b peptide

EXAMPLE 17 In-Vivo Luciferase Expression Using Microparticles WithAdsorbed Luciferase DNA

Microparticles were formed using the above-described procedures usingPLG and the detergent CTAB. Luciferase DNA was adsorbed thereon usingthe methods previously described. In vitro luciferase expression using a5 μg dose of luciferase DNA was measured using the luciferase DNA alone(1248 pg) and the microparticles with luciferase DNA adsorbed thereon(2250 pg). In vivo luciferase expression was measured in muscle on days1 and 14 following administration as follows: Two groups of mice (n=5)were each injected with either 50 μg of Luciferase plasmid or 50 μg ofPLG-CTAB-Luciferase DNA microparticles. Both groups of mice wereinjected intramuscularly in the anterior tibialis (TA) muscle on twolegs. Both TA muscles from each mouse in the two groups were harvestedeither at day 1 or day 14 and stored in a −80° C. freezer. The muscleswere ground with a mortar and pestle on dry ice. The powdered muscleswere collected in eppendorf tubes with 0.5 ml of IX Reporter LysisBuffer. The samples were vortexed for 15 minutes at room temperature.After freeze/thawing 3×, the samples were spun at 14,000 rpm for 10minutes. The supernatant of the TA muscles of each mice at eachtimepoint were pooled and 20 ul of the samples were assayed using anML3000 (Dynatech) under enhanced flash for Luciferase expression.

Luciferase determination was performed using a chemiluminiscence assay.The buffer was prepared containing 1 mg/ml of BSA in 1× Reporter Lysis(Promega). The luciferase enzyme stock (Promega) at 10 mg/ml was used asa standard, diluted to a concentration of 500 pg/20 ul. This standardwas serially diluted 1:2 down the Microlite 2 plate (Dynatech) to createa standard curve. 20 μl of the blank and the samples were also placed onthe plate and were serially diluted 1:2. The plates were placed in theML3000 where 100 ul of the Luciferase Assay Reagent (Promega) wereinjected per well. Under enhanced flash, the relative light units weremeasured for each sample.

The results are tabulated below in Table 13. TABLE 13 In vivo luciferaseIn vivo luciferase Microparticle Type expression Day 1 (pg) expressionDay 14 (pg) PLG-CTAB 9.51 44.95 Luciferase DNA adsorbed (50 μg)Luciferase DNA 6.78 9.29 alone (50 μg)

EXAMPLE 18 Immunogenicity of Microparticles with Adsorbed vs. EntrappedAntigen

Microparticles were prepared using the procedures discussed in theprevious examples. E2 protein was then adsorbed thereon as describedabove. Microparticles were also prepared with E2 entrapped therein,rather than adsorbed thereon, as described above. The microparticleswere assessed for their ability to induce IgG antibodies followingimmunization of 10 mice with each type of microparticle. The geometricmean titer (GMT) of serum from each mouse was measured, then averagedfor the group of 10 animals. Standard error (SE) was also calculated.Fisher's PLSD (significance level 5%) was measured at p=0.0006. Theresults are shown in Table 14 below: The results clearly demonstratesuperior induction of humoral immune response using the adsorbedmicroparticles of the present invention. TABLE 14 Formulation GMT SE PLGwith entrapped E2 293 270 PLG with adsorbed E2 3122 1310

EXAMPLE 19 Immunogenicity of Microparticles with HCV E1E2 ProteinAdsorbed Thereon

PLG-CTAB microparticles were prepared using the procedures discussed inthe previous examples. E1E2 protein from Hepatitis C Virus (HCV) wasadsorbed thereon. The particles were used to immunize mice, with orwithout the adjuvant Alum, in dosages of microparticles calculated toprovide either 10 μg or 100 μg of protein. Geometric mean titer wasmeasured, and the results are shown below in Table 15. TABLE 15Formulation GMT SE PLG/CTAB E1E2 (10 μg) 4117 558 PLG/CTAB E1E2 (100 μg)7583 659 PLG/CTAB E1E2 Alum (10 μg) 3356 436 PLG/CTAB E1E2 Alum (100 μg)10485 1548 HCV E1E2 DNA (10 μg) 87 63 HCV E1E2 DNA (100 μg) 7621 571

As the results indicate, the microparticles with protein adsorbedthereon produce a superior immune response at the 10 μg dose. Thisdemonstrates that the microparticles have the advantage of being usefulin eliciting immune responses at low doses where free DNA is unable togenerate such responses.

EXAMPLE 20 Immunogenicity of Microparticles with Adsorbed p24 gagprotein

PLG-PVA microparticles were prepared using the procedures discussed inthe previous examples. The protein p24 gag was then adsorbed thereon asdescribed above. The microparticles were assessed for their ability toinduce IgG, IgG1, and IgG2a antibodies following immunizations of of 10mice. The geometric mean titer (GMT) of serum collected from the mice 2weeks post 2^(nd) immunization (2wp2) and 2 weeks post 3^(rd)immunization (2wp3) were measured, then averaged for the group of 10animals. Standard error (SE) was also calculated. The results are shownin Table 16 below: The results clearly demonstrate superior induction ofhumoral immune response using the adsorbed microparticles of the presentinvention. TABLE 16 IgG IgG IgG1 IgG1 IgG2a IgG2a GMT SE GMT SE GMT SEPLG-PVA/p24 5813.59 2400.58 3741.17 2039.08 755.3 587.21 gag (2wp2) p24gag 6.6 7.91 6.51 6.85 5 1 alone (2wp2) PLG-PVA/p24 26730.29 3443.6740088.65 8989.07 6974.22 1457.74 gag (2wp3) p24 gag 7.15 5.59 8.22 12.35 1 alone (2wp3)

EXAMPLE 21 IM Immunization of p55 gag Protein and Various Adjuvants

PLG/CTAB, PLG/SDS, and PLG/PVA microparticles were formed as describedabove in the previous examples. Eight groups of microparticles were madein order to analyze the different effects of immunizing mice withadsorbed antigen p55 gag protein on microparticles vs. providing freesoluble p55 gag, and to determine the effects of having the adjuvant CpG(20 base long single stranded oligonucleotides with a CpG motif) alsoadsorbed on other microparticles or provided in free soluble form. Thedifferent groups were prepared as follows:

Group 1 used soluble p55 gag protein (recombinant HIV p55 gag proteinproduced in yeast at 2 mg/ml in tris/NaCl buffer with 2M urea) mixedwith PLG/CTAB particles with adsorbed CpG.

Group 2 used PLG/SDS particles with adsorbed p55 gag mixed with PLG/CTABparticles with adsorbed CpG.

Group 3 used PLG/SDS particles with adsorbed p55 gag mixed with freeCpG.

Group 4 used PLG/SDS particles with adsorbed p55 gag and no adjuvant.

Group 5 used PLG/PVA particles with p55 gag entrapped therein mixed withPLG/CTAB particles with CpG adsorbed.

Group 6, a control, used no antigen, and soluble CpG.

Group 7, another control, used soluble p55 gag protein and no adjuvants.

Group 8, another control, used only vaccinia virus (vv gag) expressingthe gag gene, and no adjuvants.

For each group, 10 mice were immunized with sufficient quantities ofmicroparticles or free molecules such that the dosage of p55 gag antigenand CpG adjuvant were 25 μg each (if present in the group), except forGroup 8 which was used at a dosage of 10×10⁷ pfu. The route ofimmunization was NI, except for Group 8, which route was IP. Followingimmunization, serum anti-p55 IgG titer was measured, the results ofwhich appear below in Table 17. Lysis of targets by CTL was alsomeasured with each group, the results of which appear below in Table 18.TABLE 17 Serum IgG Titer Form of p55 gag Form of CpG Serum Group ProteinAntigen Adjuvant Titer 1 soluble adsorbed on 43250 PLG/CTAB particles 2adsorbed on adsorbed on 49750 PLG/SDS particles PLG/CTAB particles 3adsorbed on soluble 62750 PLG/SDS particles 4 adsorbed on none 7550PLG/SDS particles 5 entrapped within adsorbed on 127000 PLG/PVAparticles PLG/CTAB particles 6 soluble soluble 38 7 soluble none 2913 8vaccinia virus none 938 (vv gag)

TABLE 18 PERCENT SPECIFIC LYSIS OF TARGETS Form of p55 gag Protein Formof CpG Target SvB SvB Group Antigen Adjuvant Ratio pGAG^(a) P7g+^(b) 1soluble adsorbed on 60 3 41 PLG/CTAB 15 0 15 particles 4 −1 8 2 adsorbedon adsorbed on 60 7 77 PLG/SDS PLG/CTAB 15 4 49 particles particles 4 226 3 adsorbed on soluble 60 6 51 PLG/SDS 15 3 30 particles 4 4 11 4adsorbed on none 60 4 48 PLG/SDS 15 2 21 particles 4 1 7 5 entrappedadsorbed on 60 3 37 within PLG/CTAB 15 2 17 PLG/PVA particles 4 0 4particles 6 soluble soluble 60 4 23 15 4 7 4 2 3 7 soluble none 60 1 415 −1 1 4 0 2 8 vaccinia none 60 3 52 virus 15 2 25 (vv gag) 4 3 16^(a)SvB cell line pulsed with irrelevant peptide^(b)SvB cell line pulsed with p7g peptide

EXAMPLE 22 Adsorption vs. Entrapment of p55 DNA

PLG/CTAB microparticles with adsorbed p55 DNA, and PLG/PVAmicroparticles with p55 DNA entrapped within, were formed as describedabove in the previous examples. IM immunization of mice and antibodyinduction (collection and analysis of serum) were performed as describedin the previous examples, at four weeks post 1^(st) immunization (4wp1),and 2, 4, 6, 13, and 15 weeks post 2^(nd) immunization (2wp2, 4wp2,6wp2, 13wp2, and 15wp2 respectively). The results, shown in Table 19below, demonstrate a clear advantage of the adsorbed microparticles overboth entrapped and free p55. TABLE 19 Formulation 4wp1 2wp2 4wp2 6wp213wp2 15wp2 PLG/CTAB with p55 DNA adsorbed (1 μg) 576 79300 156000227000 988000 123000 PLG/PVA with p55 DNA entrapped(1 μg) 996 1915 22151376 25100 1084 p55 plasmid alone (1 μg) 912 1149 1360 701 1075 742 p55plasmid alone (10 μg) 1489 10700 7885 26300 31600 17300

EXAMPLE 23 Microparticle Induction of Immune Response in Guinea Pigs

PLG/CTAB microparticles with adsorbed gp 120 DNA were formed asdescribed above in the previous examples. Other samples are as shownbelow in Table 20, and included the microparticles with and withoutaluminium phosphate, controls of free soluble gp120, with and withoutaluminium phosphate, and MF59 protein, encoded by gp120 DNA. IMimmunization of guinea pigs and antibody induction (collection andanalysis of serum) were performed as described in the previous examples.The results are shown in Table 20 below. TABLE 20 Formulation GMT SEPLG/CTAB gp120 adsorbed (25 μg) 1435 383 PLG/CTAB gp120 adsorbed 3624454 (25 μg) + Alum. phosphate soluble gp120 DNA (25 μg) + 119 606 Alumphosphate soluble gp120 DNA (25 μg) alone 101 55 MF59 protein (50 μg)3468 911

EXAMPLE 24 Intranasal (IN) Immunization with p55 DNA AdsorbedMicroparticles

PLG/CTAB microparticles with adsorbed p55 DNA, and PLG/DDAmicroparticles with adsorbed p55 DNA, were formed as described above inthe previous examples. IN immunization of mice with 25 or 100 μg,antibody induction (collection and analysis of serum), and CTL inductionwere performed as described in the previous examples, at two and fourweeks post 1^(st) immunization (2wp1, 4wp1), two and four weeks post2^(nd) immunization (2wp2, 4wp2), and two and four weeks post 3^(rd)immunization (2wp3, 4wp3). Controls included immunization with solublep55 DNA alone or with 10 μg cholera toxin. The results for antibodyinduction are shown in Table 21, and the results for lysis by CTL (at 4weeks post 4th immunization) are shown in Table 22 below. TABLE 21Formulation 2wp1 4wp2 2wp2 4wp2 2wp3 4wp3 PLG/CTAB with p55 DNA adsorbed(25 μg) 189 529 1412 882 908 742 PLG/CTAB with p55 DNA adsorbed (100 μg)128 383 3462 2887 289000 134000 PLG/DDA with p55 DNA adsorbed (25 μg)247 482 1223 338 940 545 PLG/DDA with p55 DNA adsorbed (100 μg) 143 13512538 1341 357000 161000 soluble p55 DNA (100 μg) + cholera toxin (10 μg)195 270 2298 617 1549 862 soluble p55 DNA (100 μg) alone 362 260 618 190285 263

TABLE 22 PERCENT SPECIFIC LYSIS OF TARGETS Dose of Target SvB SvB GroupFormulation p55 DNA Ratio pGAG^(a) P7g+^(b) 1 PLG/CTAB with 100 μg 60 −182 adsorbed p55 DNA 15 −1 53 4 12 25 2 PLG/DDA with 100 μg 60 10 47adsorbed p55 DNA 15 3 26 4 2 8 3 p55 DNA plus 100 μg 60 9 64 choleratoxin 15 2 22 (10 μg) 4 0 7 4 p55 DNA alone 100 μg 60 4 6 15 2 3 4 1 1^(a)SvB cell line pulsed with irrelevant peptide^(b)SvB cell line pulsed with p7g peptide

Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

1. A microparticle comprising: a biodegradable polymer; a detergentselected from a cationic detergent and an anionic detergent; and animmunological adjuvant, wherein said immunological adjuvant is adsorbedon the surface of said microparticle.
 2. The microparticle of claim 1,further comprising an antigen derived from a pathogenic organism or atumor, wherein said antigen is adsorbed on the surface of saidmicroparticle, encapsulated within said microparticle, or both.
 3. Themicroparticle of claim 1, wherein the biodegradable polymer is selectedfrom the group consisting of a poly(a-hydroxy acid), a polyhydroxybutyric acid, a polycaprolactone, a polyorthoester, a polyanhydride, anda polycyanoacrylate.
 4. The microparticle of claim 1, wherein themicroparticle comprises a poly(a-hydroxy acid) selected from the groupconsisting of poly(L-lactide), poly(D,L-lactide) andpoly(D,L-lactide-co-glycolide).
 5. The microparticle of claim 1, whereinthe microparticle comprises a cationic detergent.
 6. The microparticleof claim 1, wherein the microparticle comprises an anionic detergent. 7.The microparticle of claim 2, wherein the antigen is an antigencomprising a polypeptide.
 8. The microparticle of claim 2, wherein theantigen is an antigen comprising a polynucleotide.
 9. The microparticleof claim 1, wherein the microparticle further comprises an immunologicaladjuvant encapsulated within the microparticle.
 10. The microparticle ofclaim 1, wherein the immunological adjuvant is selected from a CpGoligonucleotide, an E. coli heat-labile toxin, a monophosphorylipid Acompound, and an aluminum salt.
 11. The microparticle of claim 2,wherein the microparticle comprises a cationic detergent.
 12. Themicroparticle of claim 2, wherein the microparticle comprises an anionicdetergent.
 13. A method of producing a microparticle, said methodcomprising the steps of: (a) providing an emulsion comprising (i) anorganic solvent, (ii) a biodegradable polymer, (iii) water and (iv) adetergent selected from a cationic detergent and an anionic detergent,wherein the detergent is present in the mixture at a weight to weightdetergent to polymer ratio of from about 0.00001:1 to about 0.1:1; (b)removing the organic solvent from the emulsion; and (c) adsorbing animmunological adjuvant on the surface of said microparticle.
 14. Themethod of claim 13, wherein the detergent comprises an anionicdetergent.
 15. The method of claim 13, wherein the detergent comprises acationic detergent.
 16. The method of claim 13, wherein the detergentfurther comprises a nonionic detergent.
 17. The method of claim 13,wherein the detergent is present at a weight to weight detergent topolymer ratio of from about 0.0001:1 to about 0.1:1.
 18. The method ofclaim 13, wherein the detergent is present at a weight to weightdetergent to polymer ratio of from about 0.001:1 to about 0.1:1.
 19. Themethod of claim 13, wherein the biodegradable polymer comprises apoly(a-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, apolyorthoester, a polyanhydride, or a polycyanoacrylate.
 20. The methodof claim 13, wherein the biodegradable polymer comprises apoly(a-hydroxy acid) selected from the group consisting ofpoly(L-lactide), poly(D,L-lactide) and poly(D,L-lactide-co-glycolide).21. The method of claim 13, wherein the biodegradable polymer comprisespoly(lactide-co-glycolide).
 22. The method of claim 13, wherein thepolymer is present at a concentration of about 1% to about 30% relativeto the organic solvent.
 23. The method of claim 13, wherein thebiodegradable polymer comprises poly(D,L-lactide-co-glycolide) and ispresent at a concentration of about 3% to about 10% relative to theorganic solvent.
 24. The method of claim 13, wherein said emulsion is awater-in-oil-in-water emulsion.
 25. The method of claim 13, furthercomprising providing an antigen derived from a pathogenic organism or atumor, wherein said antigen is adsorbed on the surface of saidmicroparticle, encapsulated within said microparticle, or both.
 26. Themethod of claim 25, wherein the antigen is adsorbed on the surface ofsaid microparticle.
 27. The method of claim 25, wherein the antigen isan antigen comprising a polynucleotide.
 28. The method of claim 25,wherein the antigen is an antigen comprising a polypeptide.
 29. Themethod of claim 13, further comprising providing an immunologicaladjuvant within the microparticle.
 30. A microparticle made according tothe method of claim
 13. 31. A microparticle composition comprising amicroparticle produced by the method of claim 30 and a pharmaceuticallyacceptable excipient.
 32. A method of delivering a therapeuticallyeffective amount of an immunological adjuvant to a vertebrate subjectcomprising the step of administering to the vertebrate subject amicroparticle composition of claim
 31. 33. Use of a microparticlecomposition of claim 31 for treatment of a disease.
 34. Use of amicroparticle composition of claim 31 for a vaccine.
 35. Use of amicroparticle composition of claim 31 for raising an immune response.36. The microparticle of claim 5, wherein said immunological adjuvantcomprises an immunostimulating nucleotide sequence.
 37. Themicroparticle of claim 36, wherein the immunological adjuvant comprisesa CpG oligonucleotide.
 38. The microparticle of claim 11, wherein theantigen (a) is adsorbed on the surface of the microparticle and (b)comprises a polynucleotide.
 39. The microparticle of claim 12, whereinthe antigen (a) is adsorbed on the surface of the microparticle and (b)comprises a polypeptide.
 40. The microparticle of claim 2, wherein saidantigen is selected from HIV antigens, hepatitis B virus antigens,hepatitis C virus antigens, Haemophilus influenza type B antigens,meningitis B antigens, pertussis antigens, diphtheria antigens, tetanusantigens and influenza A virus antigens.
 41. The microparticle of claim2, wherein the antigen comprises a plasmid DNA molecule.
 42. Themicroparticle of claim 1, wherein the microparticle has a diameterbetween 500 nanometers and 30 microns.
 43. The microparticle of claim 1,wherein the microparticle comprises poly(lactide-co-glycolide).
 44. Themicroparticle of claim 3, wherein the microparticle comprises an anionicdetergent.
 45. The microparticle of claim 3, wherein the microparticlecomprises a cationic detergent.
 46. A microparticle compositioncomprising a microparticle of claim 1, and a pharmaceutically acceptableexcipient.
 47. The microparticle composition claim 46, wherein saidmicroparticle composition is an injectable composition.
 48. A method ofdelivering a therapeutically effective amount of an immunologicaladjuvant to a vertebrate subject comprising the step of administering tothe vertebrate subject a microparticle composition of claim
 46. 49. Useof a microparticle composition of claim 46 for treatment of a disease.50. Use of a microparticle composition of claim 46 for a vaccine. 51.Use of a microparticle composition of claim 46 for raising an immuneresponse.
 52. A microparticle composition comprising a microparticle ofclaim 42 and a pharmaceutically acceptable excipient.
 53. Themicroparticle composition claim 52, wherein said microparticlecomposition is an injectable composition.
 54. A method of delivering atherapeutically effective amount of an immunological adjuvant to avertebrate subject comprising the step of administering to thevertebrate subject a microparticle composition of claim
 52. 55. Use of amicroparticle composition of claim 52 for treatment of a disease. 56.Use of a microparticle composition of claim 52 for a vaccine.
 57. Use ofa microparticle composition of claim 52 for raising an immune response.58. A microparticle composition comprising a microparticle of claim 43and a pharmaceutically acceptable excipient.
 59. The microparticlecomposition claim 58, wherein said microparticle composition is aninjectable composition.
 60. A method of delivering a therapeuticallyeffective amount of an immunological adjuvant to a vertebrate subjectcomprising the step of administering to the vertebrate subject amicroparticle composition of claim
 58. 61. Use of a microparticlecomposition of claim 58 for treatment of a disease.
 62. Use of amicroparticle composition of claim 58 for a vaccine.
 63. Use of amicroparticle composition of claim 58 for raising an immune response.64. A microparticle composition comprising a microparticle of claim 44and a pharmaceutically acceptable excipient.
 65. The microparticlecomposition claim 64, wherein said microparticle composition is aninjectable composition.
 66. A method of delivering a therapeuticallyeffective amount of an immunological adjuvant to a vertebrate subjectcomprising the step of administering to the vertebrate subject amicroparticle composition of claim
 64. 67. Use of a microparticlecomposition of claim 64 for treatment of a disease.
 68. Use of amicroparticle composition of claim 64 for a vaccine.
 69. Use of amicroparticle composition of claim 64 for raising an immune response.70. A microparticle composition comprising a microparticle of claim 45and a pharmaceutically acceptable excipient.
 71. The microparticlecomposition claim 70, wherein said microparticle composition is aninjectable composition.
 72. A method of delivering a therapeuticallyeffective amount of an immunological adjuvant to a vertebrate subjectcomprising the step of administering to the vertebrate subject amicroparticle composition of claim
 70. 73. Use of a microparticlecomposition of claim 70 for treatment of a disease.
 74. Use of amicroparticle composition of claim 70 for a vaccine.
 75. Use of amicroparticle composition of claim 70 for raising an immune response.